Project Summary/Abstract: Chimeric antigen receptor T (CAR-T) cell therapy has produced remarkable results in clinical trials for cancer; providing complete remissions in patients with relapsed refractory acute lymphoblastic leukemia, and offering patients a realistic hope for a cure. However, challenges related to the inability to control CAR-T cells once infused into the patient pose significant safety concerns. This includes permanent B cell aplasia and fatal cases of cytokine release syndrome. Additionally, loss of antigen expression on malignant cells renders conventional CAR-T cells ineffective against relapsed disease and has been attributed to a significant number of relapses in early stage clinical trials for ALL and CLL leukemia. Correspondingly, the development of mechanisms to control CAR-T cells represents a critical and urgent unmet medical need which warrants thorough investigation to provide a safe and efficacious CAR-T cell therapy for patients. Towards this end, this proposal describes a method of engineering antibody-based switches which enables tunable control over CAR-T cell activity. These switches mediate formation of an orthogonal immunological synapse between the target cell, switch, and CAR-T cell which is structurally, stoichiometrically, and temporally defined to enable a level of control which has not been reported previously. The long term goal of this work is to understand how these switches can improve safety and versatility in the clinic. The overall objective of this proposal is to investigate the use of switches to control activity of CAR-T cells in the context of leukemia and lymphoma using mouse models. Our central hypothesis is that antibody-based switches which control the specificity and duration of CAR-T cell activity in vivo will enable control over the efficacy, persistence, and safety of adoptively transferred CAR-T cells. The rationale for this research is that a sCAR-T cell which functions orthogonally within the patient?s immune system is safer than conventional CAR-T cell therapy because it is controlled by, and entirely dependent on, dosing of the antibody-based switch. To test the central hypothesis, aim 1 will determine the redirection of switchable CAR-T cells to more than one antigen in mouse models which mimic disease relapse. Aim 2 will determine how switch-based control effects CAR-T cell phenotype which is significant because persistent phenotypes are strongly associated with complete remissions in clinical trials. Aim 3 will use a unique mouse model which recapitulates the toxicity associated with conventional CART-19 therapy to demonstrate switch-based control over severe adverse side effects. The proposed research is a significant step in the development of a universal CAR construct which would obviate the need to reconstruct a new CAR for each antigen target. This is expected to substantially lower the cost and time of ?bench to bedside? development, as well as provide a standardized treatment regimen which is not yet possible with conventional CAR-T cells.